CN1225477C - Lung cancer early stage serum specific protein and its application - Google Patents
Lung cancer early stage serum specific protein and its application Download PDFInfo
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- CN1225477C CN1225477C CN 03116056 CN03116056A CN1225477C CN 1225477 C CN1225477 C CN 1225477C CN 03116056 CN03116056 CN 03116056 CN 03116056 A CN03116056 A CN 03116056A CN 1225477 C CN1225477 C CN 1225477C
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Abstract
The present invention discloses a new tumor marker-lung cancer early-stage serum specific protein and the purpose of the protein for diagnosing lung cancer. The present invention also provides a diagnostic reagent kit containing the lung cancer early-stage serum specific protein. The present invention can effectively detect lung cancer and can particularly detect lung cancer in early stages.
Description
Technical field
The invention belongs to technical field of biological, specifically, the present invention relates to a kind of new tumor markers lung cancer early stage blood serum special protein.The invention also discloses the purposes of lung cancer early stage blood serum special protein on diagnosing.The present invention also provides the diagnostic kit that contains the lung cancer early stage blood serum special protein.
Background technology
The primary pulmonary bronchogenic carcinoma is called for short lung cancer (Lung cancer), is to be primary in bronchial epithelial common cancers at different levels.The canceration under the stimulation of some factors of lung tissue cell makes the enlargement of lung tissue paraplasm be its pathological characteristic.Cough, pectoralgia, spitting of blood, uncomfortable in chest, out of breath, stridulate, become thin and emaciation is its main clinical manifestation.
Lung cancer has become the highest malignant tumour of mortality ratio in the worldwide.It is estimated that to the end of the year 2002, will there be the newly-increased patients with lung cancer of 169400 examples in the whole world, account for all newly-increased cancer patientss' 13%; To there be 154900 patients to die from lung cancer, account for all cancer mortality patients 28%, be higher than patient's summation far away because of mammary cancer, prostate cancer death.In China, the sickness rate of lung cancer and mortality ratio are equally also in rising trend year by year.1963～1 965, urban district, the Shanghai City lung cancer markization mortality ratio male sex was 28.5/10 ten thousand, and the women is 11.1/10 ten thousand, was 53,/10 ten thousand to male lung cancer markization sickness rate in 1999, and the women is 19/,100,000.
Lung cancer comprises small cell carcinoma (Small Cell Lung Cancer, SCLC) (account for lung cancer about 20%), non-small cell carcinoma (Non-Small Cell Lung Cancer, NSCLC) (account for lung cancer about 80%) and mixed type carcinoma.Wherein, non-small cell carcinoma is divided into squamous cell cancer (Squamous Cell Carcinoma) (25%～30%), gland cancer (Adenocarcinoma) (40%), maxicell undifferentiated carcinoma (Large-cell UndifierentiatedCarcinoma) (10%～15%) again.
Surgical operation is the preferred option of treatment lung cancer.But 65% patients with lung cancer is found and when clarifying a diagnosis, the state of an illness has been late period, has lost the opportunity of radical operation treatment, and about 80% patient is dead in back 1 year of diagnosis, so 5 years survival rates of lung cancer only are 16%.This mainly is because still lack effective lung cancer early diagnosis means at present.In recent years, flourish along with radiology, pathology and Protocols in Molecular Biology obtained many progress in the clinical diagnosis of lung cancer.
Iconography detects and comprises X line, CT scan (CT) (the CT technology such as spiral CT (the Spiral CT that comprise new development, SCT), high-resolution ct (High Resolution CT, HRCT)), the low radiation dose CT (TS-LDSCT) of thin layer, magnetic resonance imaging,MRI (MRI), positron emission scanning (PositronEmission Tomograghy, PET).
Pathology detect comprise phlegm examination of castoff cells, fiberoptic bronchoscopy, through the biopsy of skin lung puncture etc.
Molecular Biological Detection is according to the difference of clinical censorship sample, and the molecular biology index that is detected is also different.
The detectable molecular biology index of chest fluid sample comprises water-soluble cytokeratin 19 fragments (CYFRA21-1), carbohydrate antigen (CA242), tissue polypeptide antigen (TPA), neuronspecific enolase (NSE) and carcinomebryonic antigen (CEA).
The detectable molecular biology index of bronchoalveolar lavage fluid (BALF) sample comprises sudden change situation, T-antigen, the tissue polypeptide antigen (TPA) of Telomerase and telomerase hTERT genetic expression, p53, p16 and K-ras gene.
The detectable molecular biology index of sputum cast-off cells sample comprises situation, heterogeneous type cell nucleus ribonucleoprotein (heterogeneousnuclear ribonucleoprotein, hnRNP) content of crossing expression, hnRNPA2/B1 antibody of A2/B1 that sputum cast-off cells p16 gene methylation state, p16 gene extron 2 are lost.
The detectable molecular biology index of tissue sample comprises the change, Mitochondrial DNA (mtDNA) sudden change, the expression of fhit gene (FHIT) gene protein, dead related protein kinase (death-associated protein kinase, DAP) abnormal methylation of gene of the little satellite mark of karyomit(e).
The detectable molecular biology index of peripheral blood sample comprises expression level, the LUNX mRNA positive rate of peripheral blood karyocyte Keratin sulfate CK19mRNA.
The detectable molecular biology index of serum specimen comprises p53 antibody, sugar antigen-125 (CA125), carcinomebryonic antigen (CEA), serum human chorionic gonadotrophin (HCG), tumor necrosis factor-alpha (TNF-α), Tiroidina transcription factor (TTF1), mucoitin sulfate fragment (LTA).
But up to the present all correlative studys all are to be sample with the lung cancer patient, and these detect index effect in lung cancer high risk population or general population's examination and how to be still waiting further checking actually.
In addition, because lung cancer needs 10 years left and right sides time from cell generation canceration to forming clinical foci,, the lifetime of patients with lung cancer will be improved greatly if can before any pathology appears in human body even cell, not diagnose out the lung cancer omen.Yet, up to the present still lack gratifying effective lung cancer early detection means.
Therefore, this area presses for exploitation new detection of lung cancer effectively, the especially method of early detection lung cancer.
Summary of the invention
Purpose of the present invention just provides a kind of effective lung cancer early detection method and test kit.
Another object of the present invention provides relevant lung cancer early stage blood serum special protein and antibody thereof.
In a first aspect of the present invention, a kind of isolating lung cancer early stage blood serum special protein is provided, it has following feature:
(a) be present in the serum of lung cancer patient,
When (b) the laser desorption ionization flight of strengthening with the surface-time mass spectrum method (SELDI-TOF-MS) detected, its molecular weight was 4.2 ± 0.1KD, 6.3 ± 0.1KD or 7.7 ± 0.1KD.
In a preference, the condition that the laser desorption ionizationization that described surface is strengthened-time-of-flight mass spectrometry (TOFMS) detects is: use the protein biosystem PBSII of Sai Fuji company, the suitableeest laser intensity of setting be 203 and laser sensitivity be 9.
In a second aspect of the present invention, the method that whether has lung cancer early stage blood serum special protein of the present invention in a kind of test sample is provided, comprise step:
(a) get serum sample;
(b) serum sample with step (a) prepares weak anionic exchange (WCX2) protein chip;
(c) the laser desorption ionizationization of strengthening with the surface-time-of-flight mass spectrometry (TOFMS) detects, and obtains serum protein group wave spectrum;
(d) determining whether to exist molecular weight is the crest of 4.2 ± 0.1KD, 6.3 ± 0.1KD and 7.7 ± 0.1KD, exists described crest just to represent to exist described lung cancer early stage blood serum special protein.
In a preference, the condition that the laser desorption ionizationization that described surface is strengthened-time-of-flight mass spectrometry (TOFMS) detects is: use the protein biosystem PBS II of Sai Fuji company, the suitableeest laser intensity of setting be 203 and laser sensitivity be 9.
In another preference, in step (d), also comprise step: with the protein group wave spectrum of the serum sample of negative control relatively, be the crest of 4.2 ± 0.1KD, 6.3 ± 0.1KD and 7.7 ± 0.1KD thereby determine whether to exist molecular weight.
In another preference, use the protein biosystem PBSII of Sai Fuji company in the step (c).
In a third aspect of the present invention, a kind of test kit that detects lung cancer early stage blood serum special protein of the present invention is provided, it comprises:
(a) antibody of anti-described lung cancer early stage blood serum special protein.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the protein group wave spectrogram of the serum of lung cancer patient and healthy individual.
Embodiment
The inventor is through extensive and deep research, differentiates first and separated the blood serum special protein extremely relevant with lung cancer.Finished the present invention on this basis.Because having, serum draws materials conveniently, non-invasive, but and the advantage of continuous detecting, therefore the early stage biomarker of detection of lung cancer can reach a new high the early diagnosis lung cancer from serum, thereby improves the curative ratio of lung cancer and reduce the mortality ratio of lung cancer.
The core technology of proteomics research traditionally is that two-dimensional gel electrophoresis separates.The spot that shifts carries out on the glue carrying out proteic follow-up evaluation with MADLI-TOF mass spectrum and multistage LC-MS mass spectrum again behind the enzymolysis.But two-dimensional gel electrophoresis can not the big or especially little especially albumen of isolated molecule amount, because sample transfer, two-dimentional gel technique also can't separate the albumen of trace.The most important thing is that its disengaging time is long, hindered the flux of proteomics research.
In recent years, along with the appearance of multi-dimensional chromatograph isolation technique, proteomics research has been introduced novel method again.It has abandoned two-dimensional gel electrophoresis, and replaces the online connection mass spectrum of multi-dimensional chromatograph, realizes high-throughout proteome analysis.The quadrupole technology so successfully be bonded together with mass spectrum, realizes high-throughout analysis because the ability of analysis of mixtures is strong.Since 1993, the Bill Hutchens of the U.S. and Tai-Yung Yip have proposed laser desorption ionizationization--flight time mass spectrum (the Surface EnhancedLaser Desorption Ionization-time of flight Mass Spectrum of the surface reinforcement of improvement, SELDI-TOF-MS), (the ProteinChip Biosystem of protein chip system based on this technology, Ciphergen, CA USA) has had a large amount of application in Clinical Laboratory.
The ultimate principle of SELDI technology is based on the absorption of the surface reinforcement of special chip.The group of chip surface has two kinds: (as hydrophilic radical, hydrophobic grouping, the ionic group) of chemistry or biological (as metal ion, antibody, acceptor, part).Specified protein in the complicated biological sample (as cell or body fluid) is attracted on the chip by dissimilar chip surfaces.After wash-out removes weak conjugated protein, add energy absorption molecule (Energyabsorbing molecular, EAM), utilize the laser pulse radiation that combined protein is resolved and form charged ion, according to different mass-to-charge ratioes, the time that these ions fly in vacuum field is different in size, draws out a mass spectrum thus, directly information such as the molecular weight of range protein, content in the show sample.If with it and normal people or certain disease patient's collection of illustrative plates, even the collection of illustrative plates in the gene pool contrasts, and can find and catch new specific related protein matter.The advantage of SELDI technology is can high-throughput, expeditiously to analyzing without pretreated sample on a small quantity.Experimental repeatability is good, and the susceptibility height is cheap, is applicable to clinical diagnosis and large-scale crowd examination.
In the past, the research of protein involved group adopted chromatographic separation and purification, two dimensional electrophoresis, spectrum, mass spectrum quantitative and qualitative etc. to analyze the investigative technique of chemistry more.Use these means to study necessary technical qualification and require height, the instrument costliness, complex steps, consuming time tediously long, incompatible large-scale crowd's examination and Clinical Laboratory.SELDI technology and MALDI technology (the auxiliary laser desorption ionization of matrix flying time technology, (Matrix AssistedLaser Desorption Ionization-time of flight Mass Spectrum, MALDI-TOF-MS) fundamental difference is in SELDI, protein elder generation and the combination of chip surface material, add matrix, therefore the collection of illustrative plates that obtains is more single, good reproducibility, can be used for MALDI the quantification of protein analysis that can not carry out.In addition, the sample of SELDI technical Analysis does not need therefore to can be used for the biological sample of Analysis of Complex with liquid chromatography or gas-chromatography purifying in advance.Compare with 2D-PAGE (two-dimentional polyacrylamide gel electrophoresis), the advantage of SELDI technology is: the first, and the SELDI technology can be analyzed the protein that 2D-PAGE can't analyze, and comprises hydrophobic protein, the protein that the pI value is too high or too low, and low-molecular-weight protein (MW＜25kd); The second, in undressed sample, many concealed lower concentration protein can be found by the SELDI technology, have increased the chance of finding biomarker; The 3rd, the SELDI technology only needs small amount of sample, and just can obtain the result within a short period of time, and experimental repeatability is good, is fit to clinical diagnosis and extensive screening disease-related biomarker.
Therefore the SELDI technology has the qualitative analysis ability of quite high susceptibility and trace, and the early discovery to disease especially cancer is significant, than traditional detection technique advanced person many.At present, the SELDI technology is finding to have obtained a series of breakthrough developments aspect the disease marker.For example, with SELDI technical Analysis ovarian cancer patients serum, found five protein characteristic peaks (matter/lotus ratio is 534,989,2111,2251 and 2465), the sensitivity of diagnosis is 100%, and specificity is 95%, and positive predictive value is 94%.Analyze patient with breast cancer's serum with SELDI technology bond chelating Ni type protein chip, find the protein (called after NMP66) of a 28.3kD, accuracy rate of diagnosis 100%, specificity 96%.Find 5 differential protein mass peaks with the SELDI technology in the prostate cancer patients serum, these peaks of joint-detection can make the diagnostic sensitivity of prostate cancer rise to 82%, and specificity rises to 83%.With SELDI technical study transitivity bladder cancer (TCC) patient's urine sample, found that (molecular weight is 3.3～13.3kD) for 5 possible new TCC biomarkers and 7 protein cohorts.These biomarkers of coupling and the diagnosis of protein cohort can make TCC susceptibility improve and reach 87%, and specificity is 66%.With the serum of SELDI technology, find the protein of a specific expressed 13.8kD, thereby can make early screening colorectal carcinoma in conjunction with SAX2 protein chip research colorectal carcinoma patient, precancerous polyp patient.In conjunction with H4 chip research renal cell carcinoma (RCC) tissue (comprising healthy tissues, periphery cancerous tissue and central cancer tissue), find RCC excessive tissue expression 11951.2Da and 12019.9Da protein with the SELDI technology.Find RCC excessive tissue expression 12027Da protein with the SAX2 chip, but low 10205Da, 10953Da and the 12738Da protein of expressing.Thereby for patient's RCC detection provides method.(this section can be simplified.)
The appearance of SELD[technology provides an effective analysis means.Can be used for finding and the definition biomarker not only that research pharmacology is observed treatment prognosis effect at clinical medicine domain.After the present invention has determined the lung cancer early stage blood serum special protein, but just coupling SELDI technology or other technologies detect these albumen, thereby become the powerful measure of lung cancer early diagnosis and curative effect monitoring.
As used herein, term " lung cancer early stage blood serum special protein " refers to the albumen that exists in a kind of serum of lung cancer patient in early days, and when detecting with laser desorption ionization flight-time mass spectrum method (SELDI-TOF-MS) that the surface is strengthened, its molecular weight is 4.2 ± 0.1KD, 6.3 ± 0.1KD or 7.7 ± 0.1KD.
Lung cancer early stage blood serum special protein 1, lung cancer early stage blood serum special protein 2 and lung cancer early stage blood serum special protein 3 refer to that respectively molecular weight is the lung cancer early stage blood serum special protein of 4.2 ± 0.1KD, 6.3 ± 0.1KD or 7.7 ± 0.1KD.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
After having determined the lung cancer early stage blood serum special protein, the technician can separate these lung cancer early stage blood serum special proteins with ordinary method.In addition, also can produce these albumen by recombinant technology.Therefore, polypeptide of the present invention can be recombinant polypeptide, natural polypeptides, synthetic polypeptide, preferred recombinant polypeptide.Polypeptide of the present invention can be the product of natural purifying, or the product of chemosynthesis, or uses recombinant technology to produce from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Albumen of the present invention also can comprise or not comprise initial methionine residues.In addition, the present invention also comprises fragment, derivative and the analogue of people's lung cancer early stage blood serum special protein.
On the other hand, the invention still further relates to the encoding sequence of lung cancer early stage blood serum special protein, the host cell that contains the carrier of these encoding sequences and contain described carrier.Polynucleotide of the present invention can be dna form or rna form.DNA can be strand or double-stranded.
The Nucleotide full length sequence of lung cancer early stage blood serum special protein of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
On the other hand, the present invention comprises that also the lung cancer early stage blood serum special protein is had specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people's lung cancer early stage blood serum special protein or fragment.Preferably, refer to that those can combine with lung cancer early stage blood serum special protein or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Antibody of the present invention can be prepared by the known various technology of those skilled in that art.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule; Or chimeric antibody.
The antibody of anti-people's lung cancer early stage blood serum special protein can be used in the immunohistochemistry technology, detects the people's lung cancer early stage blood serum special protein in the serum specimen.
The method that whether has the lung cancer early stage blood serum special protein in a kind of test sample is to utilize the specific antibody of lung cancer early stage blood serum special protein to detect, and it comprises: serum sample is contacted with lung cancer early stage blood serum special protein specific antibody; Observe whether form antibody complex, formed antibody complex and just represented to exist in the sample lung cancer early stage blood serum special protein.
The polynucleotide of lung cancer early stage blood serum special protein also can be used for the early stage diagnosis and the treatment of lung cancer.Part or all of polynucleotide of the present invention can be used as probe stationary on microarray (microarray) or DNA chip (being called " gene chip " again), is used for analyzing the differential expression analysis and the gene diagnosis of tissue gene.Carry out RNA-polymerase chain reaction (RT-PCR) but the also transcription product of detection of lung cancer early stage blood serum special protein of amplification in vitro with the special primer of lung cancer early stage blood serum special protein.
The present invention also provides a kind of test kit that detects tumour, the primer that it contains specific amplification lung cancer early stage blood serum special protein to and/or lung cancer early stage blood serum special protein specific antibody.In addition, also can contain specific probe and/or PCR damping fluid etc.
Major advantage of the present invention is:
(a) early discovery, the early diagnosis for lung cancer provides detection method accurately and efficiently.
(b) cost is low, wants considerably cheaper than traditional diagnosis method.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
Determining of lung cancer early stage blood serum special protein
1.1 the preparation of serum
Patients with lung cancer and normal healthy controls personnel to clinical pathology is made a definite diagnosis get the 5ml peripheral blood, and behind 4 ℃ of refrigeration 2h, serum is separated out.3000g gets serum after centrifugal 15 minutes, after the packing-80 ℃ standby.Release the back use with NaAc for rare 40 times in conjunction with liquid during detection.
1.2 the preparation of chip
(Ciphergen Biosystems, Inc.) WCX2 chip are contained on the sample injector of 8-hole standby to get U.S. Sai Fuji company.
The pre-equilibration of chip:
Every hole adds 200 μ l NaAc in conjunction with liquid, hatches on the room temperature shaking table 5 minutes.Discard in conjunction with liquid, repeat 1 time.
Application of sample:
Get the serum after the dilution, every hole adds 200 μ l, hatches on the room temperature shaking table 1 hour.Discard serum.
Wash-out:
Every hole adds 200 μ l NaAC elutriants, hatches on the room temperature shaking table 5 minutes.Discard elutriant, repeat 1 time.Every hole adds 300 μ l deionized waters, after the washing, chip is pulled down in drying at room temperature from sample injector fast.
Add the energy absorption molecule (Energy Absorbed Molecular, EAM):
(Ciphergen Biosystems, Inc.), every hole adds 0.5 μ l to the fresh SPA of by specification configuration, repeats 1 time after the drying at room temperature.
1.3 serum protein group Spectrum Analysis:
To prepare chip and insert (the Ciphergen Biosystems of U.S. Sai Fuji company, Inc.) protein biosystem PBS II, choose laser intensity and be 203, laser resolution is 9, use the maximum and baseline stability in peak, write single-point routine analyzer and chip analysis program, read serum protein group wave spectrum.
1.4 the acquisition of patients with lung cancer specific proteins mass peak:
Preparation patients with lung cancer and normal healthy controls personnel sample read serum protein group wave spectrum under the same conditions on same chip.Both protein group wave spectrums are compared.
The result finds the specific expressed 4.2KD of patients with lung cancer, 6.3KD, 7.7KD protein peak as shown in Figure 1.
Embodiment 2
Utilize positive predictive value, negative predictive value, sensitivity and the specific degree of lung cancer early stage blood serum special protein detection of lung cancer
With 52 routine case serum samples and the unified numbering of 42 routine normal serum samples, adopt blind method to carry out clinical shaker test, occur in above-mentioned three characteristic peaks one promptly be judged to case.Experimental result is as follows:
| Shaker test | Lung cancer case (example) | Normal population (example) | Add up to |
| Positive (+) | 44 | 7 | 51 |
| Negative (-) | 8 | 35 | 43 |
| Add up to | 52 | 42 | 94 |
According to last table, meter sensitivity is that True Positive Rate is: 84.62%
Specific degree is that the true negative rate is: 83.33%
Positive predictive value is: 86.27%
Negative predictive value is: 81.40%
Embodiment 3
The relation of other tumours of lung cancer early stage blood serum special protein and lung and lung's inflammatory illness:
Press the identical method of embodiment 1, detect the protein group wave spectrum of the illness patients' of lung such as malignant mesothe (MM), pulmonary tuberculosis, lobar pneumonia serum, and compare.
As a result, in the patients serum of above-mentioned disease, do not find to exist simultaneously above three specific specificity albumen as yet.
Embodiment 4
The dependency of lung cancer early stage blood serum special protein and other tumours
Press the identical method of embodiment 1, detect the protein group wave spectrum of kidney, bladder cancer patient's serum, and compare.
As a result, in the patients serum of above-mentioned disease, do not find to exist simultaneously above three specific specificity albumen as yet.
Embodiment 5
The mensuration of the aminoacid sequence of lung cancer early stage blood serum special protein
Detect the American AB I QSTAR of the company LC-MS system that adopts.This is the system that a cover has engaged quadrupole and flight time mass spectrum technology, it can realize high-throughput, micro-sample, full automatic Proteomic analysis, and can identify posttranslational modification, multiple modification such as glycosylation modified, can carry out protein quantification, automatically from the beginning (de novo) order-checking.
Embodiment 6
The separation of lung cancer early stage blood serum special protein and preparation
6.1 with the serum of 20 μ l with 30 μ l 8M urea solns dilution back, 4 ℃ of mixings 10 minutes on automatic vibrator.
6.2 with 300 μ l 1M urea soln pre-equilibration Cibacron Blue posts, totally 3 times, all centrifugal 30 seconds at every turn with 1000g.
6.3 the serum sample after will diluting is added on the Cibacron Blue post.
6.4 add 50 μ l 8M urea solns dilution back, 4 ℃ of mixings 15 minutes on automatic vibrator, centrifugal 30 seconds of 1000g obtains component 1 (lung cancer early stage blood serum special protein 1).
6.5 repeating step 6.4 twice obtains component 2 (lung cancer early stage blood serum special protein 2), component 3 (lung cancer early stage blood serum special protein 3).
After 7 times of each component usefulness 1M urea soln dilutions, on the NP20 chip, to analyze, acquisition protein wave spectrogram has confirmed to have obtained its molecular weight and has been respectively 4.2KD, 6.3KD and 7.7KD, has confirmed to separate having obtained three kinds of lung cancer early stage blood serum special proteins of the present invention.
The preparation of lung cancer early stage blood serum special protein antibody
7.1 lung cancer early stage blood serum special protein 4.2 ± 0.1KD MONOCLONAL ANTIBODIES SPECIFIC FOR.
With lung cancer early stage blood serum special protein 4.2 ± 0.1KD immune mouse of separation and purification, treat that immune response occurs after, from peripheral blood, separate the B cell.Select and isolate the single lymphocyte that to secrete required antibody with haemolysis plaque analytical method.Individual cells is expanded to 1 * 10
7More than individual, extract mRNA with Quick mRNA Purification Kit.MRNA with extraction is a template, synthetic cDNA chain.With this cDNA is template, adds murine antibody variable region of heavy chain (V
H) universal primer, variable region of light chain (V
L) universal primer, carry out polymerase chain reaction, obtain the V of amplification
HGene fragment and V
LGene fragment.The capable 15g/L agarose gel electrophoresis of amplified production is identified and separated.Reclaim the V of amplification with glassmilk
HGene fragment and V
LGene fragment, with etc. the linker primer Mix of volumetric molar concentration mix, carry out polymerase chain reaction, connect V
HAnd V
LAfter the amplified production separation and purification, obtain specific single-chain antibody (ScFV).This ScFV can be used for preparation and detects required DNA chip.
These amplified production two ends are added restriction enzyme site, after purifying is quantitative, be connected to P
UC19On the carrier.To connect product transformed competence colibacillus intestinal bacteria TOP10, cut evaluation, filter out recombinant plasmid through blue hickie screening and enzyme.Form monoclonal antibody in 96 orifice plates.Filter out the monoclonal antibody strain of tiring the highest with the ELISA method, a large amount of preparations, and from culture supernatant, extract required monoclonal antibody.This monoclonal antibody can be used for preparation and detects required test kit.
7.2 lung cancer early stage blood serum special protein 6.3 ± 0.1KD and 7.7 ± 0.1KD MONOCLONAL ANTIBODIES SPECIFIC FOR are with 7.1.
The preparation of lung cancer detection test kit
Do integrated enzyme reaction with 96 orifice plates.Test kit provides 3 kinds of monoclonal antibodies at the lung cancer early stage blood serum special protein.Wrap respectively on 96 orifice plates by these three kinds of monoclonal antibodies.Standard substance, blank and sample are added 96 orifice plates and monoclonal antibody reaction.The monoclonal antibody that adds digoxigenin labeled after the washing adds the sheep anti-mouse antibody that coupling has horseradish peroxidase again.Add the OPD substrate and carry out color reaction, add 2MH
2SO
4Termination reaction.Can come the concentration of three kinds of lung cancer early stage blood serum special proteins in the quantitative sample by typical curve.And set certain threshold value and carry out clinical diagnosis.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (3)
1. isolating lung cancer early stage blood serum special protein is characterized in that, it has following feature:
(a) be present in the serum of lung cancer patient,
When (b) the laser desorption ionization flight of strengthening with the surface-time mass spectrum method detected, its molecular weight was 4.2 ± 0.1KD, 6.3 ± 0.1KD or 7.7 ± 0.1KD,
Wherein, the condition that the laser desorption ionizationization that described surface is strengthened-time-of-flight mass spectrometry (TOFMS) detects is: use the protein biosystem PBS II of Sai Fuji company, the suitableeest laser intensity of setting be 203 and laser sensitivity be 9.
2. whether there is the method for the described lung cancer early stage blood serum special protein of claim 1 in the test sample, it is characterized in that, comprise step:
(a) get people's serum sample;
(b) serum sample with step (a) prepares weak anionic exchanger matter chip;
(c) the laser desorption ionizationization of strengthening with the surface-time-of-flight mass spectrometry (TOFMS) detects, obtain serum protein group wave spectrum, wherein, the condition that the laser desorption ionizationization that described surface is strengthened-time-of-flight mass spectrometry (TOFMS) detects is: use the protein biosystem PBS II of Sai Fuji company, the suitableeest laser intensity of setting be 203 and laser sensitivity be 9;
(d) with the protein group wave spectrum of the serum sample of negative control relatively, be the crest of 4.2 ± 0.1KD, 6.3 ± 0.1KD or 7.7 ± 0.1KD thereby determine whether to exist molecular weight, exist described crest just expression have described lung cancer early stage blood serum special protein.
3. the purposes of the described lung cancer early stage blood serum special protein of claim 1 is characterized in that, is used to prepare the reagent of detection of lung cancer.
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